Salmonella typhimurium is a bacterium that can experience drastic environmental changes during its lifetime. Because of this, S. typhimurium has evolved into a metabolically versatile microorganism capable of efficient utilization of diverse environments. This versatility demands sophisticated regulatory network systems which allow S. typhimurium to adapt to environmental changes in an energy efficient manner (56,67,68,108-110). We are interested in investigating the metabolic capabilities of S. typhimurium which are relevant primarily under anaerobic growth conditions. We have focused our attention on the cobalamin (CBL, vitamin B12) biosynthetic pathway as a model system. This is a major biosynthetic pathway (approximately 30 genes), thus it represents a significant energy investment for the cell whenever this pathway Is expressed. Although de novo CBL biosynthesis in S. typhimurium only occurs anaerobically (72), we have shown that under certain growth conditions, the cobalamin biosynthetic (cob) genes can be highly transcribed in the presence of high oxygen levels. Despite this high transcription, CBL is not synthesized. This could be due to oxygen intermediates or enzymes, or it may suggests the existence of sites of oxygen regulation at the enzyme level. We propose to investigate three unique aspects of cobalamin biosynthesis in S. typhimurium. 1. Role of the CobK protein in the conversion of 4,5- dimethylphenylenediamine (DMPDA) to 5,6-dimethylbenzimidazole (DMB). We believe that addition of a one-carbon unit to DMPDA is the terminal step in DMB biosynthesis. The metabolic origin of the C1 unit will be determined, the CobK will be purified and biochemically characterized, and its role in DMPDA conversion to DMB established. 2. Role of the CobU protein in the assembly of the nucleotide loop of cobalamin, and in the assimilation of incomplete, exogenous corrinoids. CobU is a guanylyltransferase required for the synthesis of adenosyl- cobinamide-GDP. We have uncovered a unique adenosyltransferase activity associated with CobU that is important for the assimilation of incomplete, exogenous corrinoids. This activity is only observed under anaerobic conditions. The guanylyltransferase and adenosyltransferase activities of wild-type CobU will be characterized in vitro, and compared to specific mutant proteins presumed inactive for only one of the activities. Regions of CobU necessary for each activity will be identified. 3. Role of the CobA protein in the adenosylation of the corrin macrocycle. Corrinoid adenosylation requires three steps, two reductions and one adenosyl transfer. cobA mutants are defective in attaching the adenosyl ligand to the corrin ring, but the precise role of the CobA protein in this process is unclear. We will characterize the CobA gene product, and identify the intermediate in de novo corrin ring intermediate that accumulates in cobA mutants.